A lithium-ion-active aerolysin nanopore for effectively trapping long single-stranded DNA

Area of Science:

• Nanopore sensing
• Biophysics
• Molecular diagnostics

Background:

• Wild-type aerolysin (AeL) nanopores enable label-free single nucleotide discrimination of short oligonucleotides (≤10 nt).
• A key limitation of AeL is its insufficient capture ability for long single-stranded DNA (>10 nt).

Purpose of the Study:

• To develop a novel strategy for enhancing the capture of long single-stranded DNA (ssDNA) by aerolysin (AeL) nanopores.
• To demonstrate AeL's capability for detecting ssDNA longer than 100 nt using the new method.

Main Methods:

• Electrostatic focusing of long ssDNA into a lithium-chloride (LiCl)-activated AeL nanopore.
• Comparative analysis of LiCl versus potassium chloride (KCl) for ssDNA capture efficiency.
• Computational calculations and molecular dynamics simulations to elucidate the mechanism of enhanced capture.

Main Results:

• The novel strategy successfully enabled AeL detection of ssDNA longer than 100 nt for the first time.
• LiCl significantly improved AeL's capture ability for 60 nt ssDNA by 2.63- to 10.23-fold compared to KCl.
• Molecular dynamics simulations indicated that Li+ binding neutralizes AeL, reducing the energy barrier for ssDNA capture.

Conclusions:

• The LiCl-based electrostatic focusing strategy overcomes the limitation of AeL's poor capture of long ssDNA.
• This advancement significantly enhances AeL nanopore performance for detecting longer DNA molecules.
• The findings pave the way for high-throughput applications of AeL in genetic and epigenetic diagnostics.